125I-Spiperone: a novel ligand for D2 dopamine receptors

125I-Spiperone binds with high affinity (KD 0.3 nM) to a single specific site (Bmax 34 pmol/g wet weight) in homogenates of rat corpus striatum. Specific binding is about 40-60 percent of total binding and is displaced stereo-specifically by butaclamol and clopenthixol. Neuroleptic drugs of various classes are potent inhibitors of 125I-spiperone binding (Ki's 1-10 nM). Selective dopamine antagonists such as sulpiride (Ki 50 nM) and dopamine agonists such as apomorphine (Ki 200 nM) are also potent inhibitors. The drug specificity of 125I-spiperone binding correlates well with that of 3H-spiperone binding, providing good evidence that 125I-spiperone labels D2 dopamine receptors in striatal membranes. 125I-Spiperone, with its high specific activity (2200 Ci/mmol) may prove to be a useful ligand in studies examining D2 dopamine receptors in soluble preparations and by autoradiography. Furthermore iodinated spiperone may be useful in radioreceptor assays of neuroleptic drug levels and, in a 123I-labeled form, for imaging of dopamine receptors, in vivo, using single photon tomography.     

Iodotyrosylation of peptides using tertiary-butyloxycarbonyl-l-[125I]iodotyrosine N-hydroxysuccinimide ester

A rapid method for the iodotyrosylation of peptides using tertiary-butyloxycarbonyl-l-[¹²⁵I]-iodotyrosine N-hydroxysuccinimide ester is described. The method, like that of Bolton and Hunter (1973, Biochem. J.133, 529–539), uses nonoxidizing conditions, is applicable to tyrosine-free peptides, and results in an easily isolated product with decreased cationic charge. The present method has the additional advantage that the derivatized peptide is readily deblocked to form a radiolabeled product with the same net charge as the starting material.     

High affinity dopamine D2 receptor radioligands. 2. [125I]epidepride, a potent and specific radioligand for the characterization of striatal and extrastriatal dopamine D2 receptors.

Epidepride, (S)-N-[(1-ethyl-2-pyrrolidinyl)methyl]-5-iodo-2,3-dimethoxybenzamide+ ++, the iodine analogue of isoremoxipride (FLB 457), was found to be a very potent dopamine D2 receptor antagonist. Optimal in vitro binding required incubation at 25 degrees C for 4 h at pH 7.4 in a buffer containing 120 mM NaCl, 5 mM KCl, 2 mM CaCl2 and 1 mM MgCl2. Scatchard analysis of in vitro binding to striatal, medial frontal cortical, hippocampal and cerebellar membranes revealed a KD of 24 pM in all regions, with Bmax's of 36.7, 1.04, 0.85, and 0.37 pmol/g tissue, respectively. The Hill coefficients ranged from 0.91-1.00 in all four regions. The IC50's for inhibition of [125I]epidepride binding to striatal, medial frontal cortical, and hippocampal membranes for SCH 23390, SKF 83566, serotonin, ketanserin, mianserin, naloxone, QNB, prasozin, clonidine, alprenolol, and norepinephrine ranged from 1 microM to greater than 10 microM. Partial displacement of [125I]epidepride by nanomolar concentrations of clonidine was noted in the frontal cortex and hippocampus, but not in the striatum. Scatchard analysis of epidepride binding to alpha 2 noradrenergic receptors in the frontal cortex and hippocampus revealed an apparent KD of 9 nM. At an epidepride concentration equal to the KD for the D2 receptor, i.e. 25 pM, no striatal alpha 2 binding was seen and only 7% of the specific epidepride binding in the cortex or hippocampus was due to binding at the alpha 2 site. Correlation of inhibition of [3H]spiperone and [125I]epidepride binding to striatal membranes by a variety of D2 ligands revealed a correlation coefficient of 0.99, indicating that epidepride labels a D2 site. In vitro autoradiography revealed high densities of receptor binding in layers V and VI of prefrontal and cingulate cortices as well as in striatum. In vivo rat brain uptake revealed a hippocampal:cerebellar and frontal cortical:cerebellar ratio of 2.2:1 which fell to 1.1:1 following haloperidol pretreatment. These properties suggest that [125I]epidepride is a superior radioligand for the in vitro and in vivo study of striatal and extrastriatal dopamine D2 receptors.     

[125I]LSD binding to serotonin and dopamine receptors in bovine caudate membranes

[¹²⁵I]LSD (labeled at the 2 position) has been introduced as the first ¹²⁵I-labeled ligand for serotonin 5-HT₂ (S2) receptors. In the present study we examined the binding of [¹²⁵I]LSD and its non-radioactive homologue, 2I-LSD, to bovine caudate homogenates. The binding of [¹²⁵I]LSD is saturable, reversible, stereospecific and is destroyed by boiling the membranes. The specific to total binding ratio in this tissue is 75–80% and Scatchard plots of the binding data reveal K_d = 1.1 nM, B_max = 9.6 fmol/mg wet weight tissue. The association and dissociation rate constants are highly temperature dependent. At 0°C the net dissociation is less than 5% after 1 h and the association rate is proportionately slow. IC₅₀ values for a variety of compounds show a clear 5-HT₂ (S2) serotonergic pattern at this [¹²⁵I]LSD site. Blockage of this primary 5-HT₂ (S2) caudate binding site by 0.3 μM mianserin reveals the presence of a weaker [¹²⁵I]LSD binding site with a K_d = 9.1 nM, B_max = 7.6 fmol/mg tissue. This secondary site is a D3 dopaminergic receptor site, as shown by the relative abilities of various displacers to inhibit this binding. Binding studies with nonradioactive 2I-LSD reveal a clear preference for D2 over D3 dopamine receptor sites. [¹²⁵I]LSD is a sensitive and selective label for 5-HT₂ (S2) serotonin receptor sites in both rat frontal cortex and bovine caudate membranes. Blockage of the primary bovine caudate [¹²⁵I]LSD binding site with mianserin allows the high sensitivity of [¹²⁵I]LSD to be applied to D2 dopamine receptor studies as well.     

[3H]Mesulergine,a selective ligand for serotonin-2 receptors

[3H]Mesulergine binds with high affinity to rat cerebral cortex membranes. (K_D = 1.9 nM, B_max = 11.3 pM/g tissue). The binding of this ligand is selective for serotonin-2 receptors: Its next highest affinity, which is for dopamine receptors labelled by neuroleptics, is about 50 times weaker than its affinity for serotonin-2 receptors. No significant affinity for serotonin-1, α₁-adrenergic or histamine H1 receptors was observed.Specific [3H]mesulergine binding was diminished in the presence of low concentrations of lithium ions.     

A novel radioligand [125I]BQ-3020 selective for endothelin (ETB) receptors.

A linear endothelin (ET) analog, N-acetyl-LeuMetAspLysGluAlaValTyrPheAlaHisLeu-AspIleIleTrp (BQ-3020), is highly selective for ETB receptors. BQ-3020 displaces [125I]ET-1 binding to ETB receptors (nonselective to ET isopeptides) in porcine cerebellar membranes (IC50: 0.2nM) at a concentration 4,700 times lower than that to ETA receptors (selective to ET-1) on aortic vascular smooth muscle cells (VSMC) (IC50: 940nM). BQ-3020 as well as ET-1 and ET-3 elicits vasoconstriction in the rabbit pulmonary artery. The ETA antagonist BQ-123 failed to inhibit this BQ-3020-induced vasoconstriction. Furthermore, BQ-3020 elicits endothelium-dependent vasodilation. These data indicate that BQ-3020 has ETB agonistic activity. The radioligand [125I]BQ-3020 binds to cerebellar membranes at single high affinity sites (Kd = 34.4pM), whereas it scarcely binds to VSMC. [125I]BQ-3020 binding to the cerebellum was displaced by BQ-3020, ET-1 and ET-3 in a nonselective manner (IC50: 0.07-0.17nM). However, the binding of [125I]BQ-3020 was insensitive to the ETA antagonist BQ-123 and other bioactive peptides. Both [125I]ET-1 and [125I]BQ-3020 show slow onset and offset binding kinetics to ETB receptors. These data indicate that the radioligand [125I]BQ-3020 selectively labels ETB receptors and that the slow binding kinetics of ET-1 are dependent on the peptide sequence from Leu6 to Trp21, but not on the structure formed by its two disulfide bridges.     

Photoaffinity labelling of dopamine D2 receptors by [3H]azidomethylspiperone

We have characterized the dopamine D2 receptor photoaffinity probe, [3H]azido-N-methylspiperone ([3H]AMS). In the absence of light, [3H]AMS bound reversibly and with high affinity (Kd 70 pM) to sites in canine striatal membranes and was competitively inhibited by dopaminergic agonists and antagonists with an appropriate D2 receptor specificity. Upon photolysis, [3H]AMS covalently incorporated into a peptide of Mr 92,000 as assessed by fluorography following SDS-polyacrylamide gel electrophoresis. Labelling of this peptide was specifically and stereoselectively blocked by D2 antagonists and agonists. Minor specifically labelled peptides of Mr 70,000-55,000 were observed under some conditions and were the result of proteolytic degradation of the peptide at Mr 92,000.     

125]I-spectramide: a novel benzamide displaying potent and selective effects at the D2 dopamine receptor

The new substituted benzamide Spectramide, (N-[2-[4-iodobenzyl-N-methylamino]-2-methoxy-4-ethyl]-5-chloro- methylamine] benzamide) labelled with 125I was used as a potent and highly selective dopamine-D2 receptor antagonist in rat striatal homogenates for in vitro receptor binding. Kinetic experiments demonstrated the reversibility of the binding and the estimated Kd from saturation analysis was 25 pM, with a Bmax of 20 pmol/g of tissue. Competition studies showed that spectramide did not interact potently with the D1 or dopamine-uptake site. Drugs known to interact with other receptor systems were weak competitors of the binding, while binding was potently inhibited by other D2 antagonists, such as spiperone and eticlopride. These data indicate that Spectramide binds selectively and with high affinity to the dopamine D2 receptors, and may prove to be a useful tool for the study of these receptors in vivo using PET or SPECT.     

Identification of a 2-phenyl-substituted octahydrobenzo[f]quinoline as a dopamine D3 receptor-selective full agonist ligand

This work describes the identification of a novel class of octahydrobenzo[f]quinolines as dopamine D₃-selective full agonists. We developed a facile method that utilizes Suzuki coupling for easy incorporations of various substituted pendant rings into the scaffold. A small focused library of octahydrobenzo[f]quinolines 5 was synthesized, and these compounds demonstrated at least 14-fold D₂-like selectivity over D₁ in native porcine striatal tissue. Furthermore, n-propyl analog 5f was found to be a high affinity (K_i = 1.1 nM) D₃ dopamine full agonist with 145-fold selectivity over the D₂ receptor and about 840-fold selectivity over the D₁ receptor.     

In vitro binding properties and autoradiographic imaging of 3-iodobenzamide ([125I]-IBZM): a potential imaging ligand for D-2 dopamine receptors in SPECT

The in vitro binding properties of the [125I] labeled benzamide (S(-)-N-[(1-ethyl-2-pyrrolidinyl)-methyl]-2-hydroxy-3-iodo-6-methoxy- benzamide, IBZM) were determined in bovine and mouse caudate membrane homogenates and by autoradiography of mouse brain slices. [125I]-IBZM binding is saturable and reversible with a Bmax of 373 +/- 51 fmol/mg protein and a Kd of 3.1 +/- 0.62 nM (mean +/- SD, Scatchard analyses) and 0.56 nM as calculated by association and dissociation time constants. In competition experiments, Ki values for the D-2 antagonists YM-09151-2 and spiperone are 4 orders of magnitude lower than the Ki value for the D-1 antagonist SCH-23390 and S(-)-IBZM is ten-fold more potent than R(+)-IBZM. [125I]-IBZM has a low affinity for serotonin S-2 and for alpha receptors. Therefore, it is a highly selective ligand for dopamine D-2 receptors. Autoradiographic images of brain sections incubated with [125I]-IBZM show the dopamine D-2 receptors of the striatum, nucleus accumbens and olfactory tubercle with a high ratio of specific to nonspecific binding. Thus, S(-)-IBZM, when labeled with [123I], may be useful for in vivo imaging of dopamine D-2 receptors by single photon emission computerized tomography (SPECT).     

Interaction of iodinated human [D-Ala2]beta-endorphin with opitate receptors.

The interaction of beta-endorphin with opiate receptors was studied by using the radioiodinated, metabolically stable D-Ala2 derivative of human beta-endorphin. This analog binds specifically to rat brain membrane preparations with an apparent Kd of about 2.5 x 10-9 M. The ability of various enkephalin analogs, as well as opiate agonists and antagonists, to inhibit the binding of beta-endorphin clearly demonstrates that this peptide can bind to opiate receptors. However, the effects of various cations on the binding of 125I-[D-Ala2]beta-endorphin are markedly different from those found for enkephalin binding. Sodium ion at physiological concentrations decreases substantially the binding of enkephalins but only slightly decreases endorphin binding, whereas manganese enhances enkephalin binding but has no effect on endorphin binding. Moreover, potassium (100 mM) decreases the binding of beta-endorphin but does not affect enkephalin binding. These results suggest that beta-endorphin and enkephalin bind differently to the same receptor or bind to different receptors with overlapping specificity.     

Pharmacological evaluation of (+)-2- [123I]A-69024: a radioligand for in vivo studies of dopamine D1 receptors

(+)-2-[123I] A-69024, [(+)-1-(2-[123I] iodo-4,5-dimethoxy-benzyl)-7-hydroxy-6-methoxy-2-methyl-1,2,3,4-tetrahydroisoquinoline], is a specific and enantioselective dopamine D1 receptor radioligand. The in vivo biodistribution of this radioligand in rats showed high brain uptake and a distribution consistent with the density of dopamine D1 receptors. Highest uptake was observed in the striatum (0.65 %ID/g) at 5 min followed by clearance. As a measure of specificity the striatum/cerebellar ratio reached a maximum of 3.9 at 30 min post-injection. Radioactivity in the striatum was reduced by 68% by pre-administration of the D1 antagonist SCH 23390. Pre-administration of other dopamine binding drugs, spiperone (D2), 7-OH-DPAT (D3), and clozapine (D4) displayed no inhibitory effect on (+)-2-[123I]A-69024 accumulation in any brain region. Ketanserin (5-HT2/5-HT2C) and haloperidol (D2 receptor antagonist/sigma receptor ligand) also displayed no inhibitory effect in any brain region studied. With the pharmacologically inactive enantiomer, (-)-2-[123I]A-69024, the brain uptake was determined to be non-specific since a striatum/cerebellar ratio of near 1 was observed throughout the time course of the experiment. (+)-2-[123I]A-69024 displays enantioselectivity for dopamine D1 receptors and may deserve further investigation as a possible SPECT radioligand.     

4-[123I]Iodospiperone as a ligand for dopamine DA receptors: In vitro and in vivo experiments in a rat model

Radioiodinated spiperone is of interest for dopamine (DA) receptor studies in the living human brain by single photon emission computed tomography (SPECT). Stimulated by data obtained with [¹¹C]-N-methyl-spiperone we synthesized 4-[¹²³I]iodospiperone and investigated the in vitro binding characteristics of this ligand to the striatal membrane of the rat and the in vivo distribution over various rat brain regions. The in vitro binding experiments showed that this radioligand displays about 10 times less affinity for the DA receptor than spiperone and specific binding, as shown with [³H]spiperone, was not observed. Displacement by butaclamol was not observed. The in vivo studies demonstrated that both 4-[¹²³I]iodospiperone and [³H]spiperone concentrate in striatal tissue, respectively, 1.9 and 3.5 times as high as in cerebellar tissue.Haloperidol pretreatment largely prevented this accumulation. In view of the obtained target-to-non-target ratios we believe, however, that this accumulation in brain areas rich in DA-receptors does not offer prospects for clinical receptor imaging with SPECT.     

125I][D-Ala2]deltorphin-I: a high affinity, delta-selective opioid receptor ligand.

The selective delta opioid agonist [D-Ala2]deltorphin-I was radioiodinated and the product purified using reverse phase HPLC. The binding characteristics and distribution profile of [125I][D-Ala2]deltorphin-I were assessed in mouse brain using homogenate binding techniques and quantitative autoradiography. [125I][D-Ala2]deltorphin-I bound with high affinity to a single class of sites (KD = 0.5 nM) in brain membrane preparations and striatal sections. Competition studies indicated that [125I][D-Ala2]deltorphin-I was selectively labeling delta opioid receptors as shown by the ratio of apparent affinities for mu and delta receptors (KI mu/KI delta = 1388). The autoradiographical distribution profile of [125I][D-Ala2]deltorphin-I binding sites was also consistent with that of other delta-selective radioligands. The data indicate that [125I][D-Ala2]deltorphin-I binds to delta opioid receptors with high affinity and selectivity. Because of its very high specific activity, it can be detected rapidly with high sensitivity by autoradiographic emulsion.     

Autoradiographic localizatio of [125I]-C-ANP compared to [125I]-ANP in rat tissue

Summary Whole-body autoradiography demonstrated the different distribution of [¹²⁵I]-C-ANP and [¹²⁵I]-ANP to rat tissues. Highest enrichment of radioactivity of both labelled peptides was found in the kidney. In some organs we found remarkable differences between [¹²⁵I]-ANP and [¹²⁵I]-C-ANP. In the kidney cortex, especially in the glomeruli, as well as in the endocardium, the zona glomerulosa and the medulla of the adrenal gland, where high levels of radioactivity after [¹²⁵I]-ANP administration were detected, no or just few radioactivity was found after administration of [¹²⁵I]-C-ANP. On the other hand in the kidney papilla and the outer subcortical medulla, characteristic blackening was found after [¹²⁵I]-C-ANP administration. Those differences might be important for the understanding of pharmacological actions of ANP analogues.This work is part of the doctoral thesis of Frank Heidemann to be presented at the Ludwig-Maximilians-Universität München, FRG     

Interactions of GIPC with dopamine D2, D3 but not D4 receptors define a novel mode of regulation of G protein-coupled receptors

The C-terminus domain of G protein-coupled receptors confers a functional cytoplasmic interface involved in protein association. By screening a rat brain cDNA library using the yeast two-hybrid system with the C-terminus domain of the dopamine D(3) receptor (D(3)R) as bait, we characterized a new interaction with the PDZ domain-containing protein, GIPC (GAIP interacting protein, C terminus). This interaction was specific for the dopamine D(2) receptor (D(2)R) and D(3)R, but not for the dopamine D(4) receptor (D(4)R) subtype. Pull-down and affinity chromatography assays confirmed this interaction with recombinant and endogenous proteins. Both GIPC mRNA and protein are widely expressed in rat brain and together with the D(3)R in neurons of the islands of Calleja at plasma membranes and in vesicles. GIPC reduced D(3)R signaling, cointernalized with D(2)R and D(3)R, and sequestered receptors in sorting vesicles to prevent their lysosomal degradation. Through its dimerization, GIPC acts as a selective scaffold protein to assist receptor functions. Our results suggest a novel function for GIPC in the maintenance, trafficking, and signaling of GPCRs.     

Probing the salmeterol binding site on the beta 2-adrenergic receptor using a novel photoaffinity ligand, [(125)I]iodoazidosalmeterol.

Salmeterol is a long-acting beta2-adrenergic receptor (beta 2AR) agonist used clinically to treat asthma. In addition to binding at the active agonist site, it has been proposed that salmeterol also binds with very high affinity at a second site, termed the "exosite", and that this exosite contributes to the long duration of action of salmeterol. To determine the position of the phenyl ring of the aralkyloxyalkyl side chain of salmeterol in the beta 2AR binding site, we designed and synthesized the agonist photoaffinity label [(125)I]iodoazidosalmeterol ([125I]IAS). In direct adenylyl cyclase activation, in effects on adenylyl cyclase after pretreatment of intact cells, and in guinea pig tracheal relaxation assays, IAS and the parent drug salmeterol behave essentially the same. Significantly, the photoreactive azide of IAS is positioned on the phenyl ring at the end of the molecule which is thought to be involved in exosite binding. Carrier-free radioiodinated [125I]IAS was used to photolabel epitope-tagged human beta 2AR in membranes prepared from stably transfected HEK 293 cells. Labeling with [(125)I]IAS was blocked by 10 microM (-)-alprenolol and inhibited by addition of GTP gamma S, and [125I]IAS migrated at the same position on an SDS-PAGE gel as the beta 2AR labeled by the antagonist photoaffinity label [125I]iodoazidobenzylpindolol ([125I]IABP). The labeled receptor was purified on a nickel affinity column and cleaved with factor Xa protease at a specific sequence in the large loop between transmembrane segments 5 and 6, yielding two peptides. While the control antagonist photoaffinity label [125I]IABP labeled both the large N-terminal fragment [containing transmembranes (TMs) 1-5] and the smaller C-terminal fragment (containing TMs 6 and 7), essentially all of the [125I]IAS labeling was on the smaller C-terminal peptide containing TMs 6 and 7. This direct biochemical evidence demonstrates that when salmeterol binds to the receptor, its hydrophobic aryloxyalkyl tail is positioned near TM 6 and/or TM 7. A model of IAS binding to the beta 2AR is proposed.     

Autoradiographic localization of [125I]-C-ANP compared to [125I]-ANP in rat tissue.

Whole-body autoradiography demonstrated the different distribution of [125I]-C-ANP and [125I]-ANP to rat tissues. Highest enrichment of radioactivity of both labelled peptides was found in the kidney. In some organs we found remarkable differences between [125I]-ANP and [125I]-C-ANP. In the kidney cortex, especially in the glomeruli, as well as in the endocardium, the zona glomerulosa and the medulla of the adrenal gland, where high levels of radioactivity after [125I]-ANP administration were detected, no or just few radioactivity was found after administration of [125I]-C-ANP. On the other hand in the kidney papilla and the outer subcortical medulla, characteristic blackening was found after [125I]-C-ANP administration. Those differences might be important for the understanding of pharmacological actions of ANP analogues.     

Synthesis of novel lactam derivatives and their evaluation as ligands for the dopamine receptors, leading to a D(4)-selective ligand

The preparation of some lactam (cyclic amide) derivatives bearing various phenylpiperazinylbutyl side chains attached to the amide nitrogen together with their dopamine receptor affinity study is described. The synthesis of the target compounds involved the preparation of the intermediate bromobutyl derivatives of the appropriate lactam followed by N-alkylation of the appropriate phenylpiperazines with these intermediates. Radioligand binding studies at D(2)-D(5) receptor subtypes and a functional calcium assay of the target compounds at D(2) and D(5) receptor subtypes were performed. All compounds, except 12a and 12b, showed selectivity towards the D(2)-like receptor subtypes. Selectivity of the indolinone derivatives 11a-d at the D(4) receptors was observed. Compound 11b exhibited a remarkable affinity to hD(4) receptors with K(i) value of 0.04+/-0.02 nm and was >43,000-fold selective over the hD(2) receptor. In the functional assay, all the active compounds were of antagonistic activity.     

Benzamides derived from 1,2-diaminocyclopropane as novel ligands for human D2 and D3 dopamine receptors

Benzamides (3a-f) derived from 4-amino-5-chloro-2-methoxybenzoic acid and either cis or trans 1,2-diaminocyclopropane were synthesised and were evaluated in binding assays employing, bovine striatal D2 receptors, recombinant human hD2 and hD3 receptors expressed in CHO cells and rat, cortical 5-HT3 and striatal 5-HT4 receptors. The cis and trans isomers of the derivatives were isolated and characterised. The results demonstrated the superiority of the cis conformers over the trans conformers in dopamine receptor binding assays (Ki hD2 = 13.4 and 6.9 nM and Ki hD3 = 17.7 and 4.5 nM for the cis-3b and cis-3f compounds, respectively; Ki hD2 = 816 and >l000 nM and Ki hD3 = 469 and >1000 nM for the corresponding trans-3b and trans-3f compounds respectively). The cis compounds are folded: the benzamide group and the basic nitrogen atom were in a syn relationship. Compound 3f can be superimposed with a conformation of the tropane derivative, BRL 25594, having the benzyl group in an axial position to give a suitable fit, indicating that both compounds may have a common binding site in the dopamine receptor.